Advertisement

Acute Oxidative Stress Can Reverse Insulin Resistance by Inactivation of Cytoplasmic JNK*

Open AccessPublished:April 29, 2010DOI:https://doi.org/10.1074/jbc.M109.093633
      Chronic oxidative stress results in decreased responsiveness to insulin, eventually leading to diabetes and cardiovascular disease. Activation of the JNK signaling pathway can mediate many of the effects of stress on insulin resistance through inhibitory phosphorylation of insulin receptor substrate 1. By contrast, exercise, which acutely increases oxidative stress in the muscle, improves insulin sensitivity and glucose tolerance in patients with Type 2 diabetes. To elucidate the mechanism underlying the contrasting effects of acute versus chronic oxidative stress on insulin sensitivity, we used a cellular model of insulin-resistant muscle to induce either chronic or acute oxidative stress and investigate their effects on insulin and JNK signaling. Chronic oxidative stress resulted in increased levels of phosphorylated (activated) JNK in the cytoplasm, whereas acute oxidative stress led to redistribution of JNK-specific phosphatase MKP7 from the nucleus into the cytoplasm, reduction in cytoplasmic phospho-JNK, and a concurrent accumulation of phospho-JNK in the nucleus. Acute oxidative stress restored normal insulin sensitivity and glucose uptake in insulin-resistant muscle cells, and this effect was dependent on MKP7. We propose that the contrasting effects of acute and chronic stress on insulin sensitivity are driven by changes in subcellular distribution of MKP7 and activated JNK.

      Introduction

      Chronic oxidative stress is one of the major sources of metabolic abnormalities associated with Type 2 diabetes (
      • Demirbag R.
      • Yilmaz R.
      • Gur M.
      • Celik H.
      • Guzel S.
      • Selek S.
      • Kocyigit A.
      ,
      • Rehman A.
      • Nourooz-Zadeh J.
      • Möller W.
      • Tritschler H.
      • Pereira P.
      • Halliwell B.
      ,
      • Song F.
      • Jia W.
      • Yao Y.
      • Hu Y.
      • Lei L.
      • Lin J.
      • Sun X.
      • Liu L.
      ). High glucose and fatty acid levels lead to increased production of reactive oxygen species (ROS),
      The abbreviations used are: ROS
      reactive oxygen species
      MAPK
      mitogen-activated protein kinase
      JNK
      Jun N-terminal kinases
      IRS1
      insulin receptor substrate 1
      DMEM
      Dulbecco's modified Eagle's medium
      MKP7
      MAP kinase phosphatase 7
      siRNA
      small interfering RNA.
      which can cause insulin resistance in peripheral metabolic tissues. This leads to decreased glucose uptake in muscle and adipose tissue, and eventually, pancreatic β cell failure, glucose intolerance, and frank diabetes (
      • Muellenbach E.A.
      • Diehl C.J.
      • Teachey M.K.
      • Lindborg K.A.
      • Archuleta T.L.
      • Harrell N.B.
      • Andersen G.
      • Somoza V.
      • Hasselwander O.
      • Matuschek M.
      • Henriksen E.J.
      ,
      • Gao L.
      • Laude K.
      • Cai H.
      ,
      • Björnholm M.
      • Zierath J.R.
      ,
      • Evans J.L.
      • Maddux B.A.
      • Goldfine I.D.
      ,
      • Houstis N.
      • Rosen E.D.
      • Lander E.S.
      ).
      The mechanistic link between increased ROS levels and insulin resistance is activation of several signaling pathways, primarily mitogen-activated protein kinases (MAPK) pathways. JNK (Jun N-terminal kinases) are MAP kinases activated by cellular stresses, including oxidative stress, and play a role in apoptosis and survival, stress resistance, and immune response (
      • Raman M.
      • Chen W.
      • Cobb M.H.
      ). Upstream signaling leading to JNK activation involves stress-induced MAPK kinases MEKK4 and MEKK7, as well as scaffold protein JIP (JNK-interacting protein) (
      • Wang X.
      • Destrument A.
      • Tournier C.
      ). Activation of JNK leads to dimerization followed by translocation into the nucleus, where it can phosphorylate its downstream target c-Jun, leading to activation of stress response and apoptotic pathways. JNKs are specifically dephosphorylated and inactivated by MAP kinase phosphatase 7 (MKP7), which also acts as a shuttle protein and was proposed to be involved in JNK nucleocytoplasmic translocation (
      • Masuda K.
      • Shima H.
      • Watanabe M.
      • Kikuchi K.
      ).
      Obesity increases JNK activation in muscle and adipose tissue in mice. Genetic ablation or pharmacological inhibition of JNK results in marked improvement of insulin sensitivity in mouse models of diet-induced obesity and insulin resistance (
      • Björnholm M.
      • Zierath J.R.
      ,
      • Bloch-Damti A.
      • Potashnik R.
      • Gual P.
      • Le Marchand-Brustel Y.
      • Tanti J.D.
      • Rudich A.
      • Bashan N.
      ,
      • Hirosumi J.
      • Tuncman G.
      • Chang L.
      • Görgün C.Z.
      • Uysal K.T.
      • Maeda K.
      • Karin M.
      • Hotamisligil G.S.
      ). Mechanistically, JNK has been shown to phosphorylate IRS1 (insulin receptor substrate 1) at multiple serine residues, targeting IRS1 to degradation by the proteasome machinery (
      • Bloch-Damti A.
      • Potashnik R.
      • Gual P.
      • Le Marchand-Brustel Y.
      • Tanti J.D.
      • Rudich A.
      • Bashan N.
      ). Inhibition of JNK activation prevents IRS1 degradation and promotes downstream insulin signaling and insulin-dependent glucose uptake. JNK activation is a key mediator of ROS-induced insulin resistance (
      • Lee Y.H.
      • Giraud J.
      • Davis R.J.
      • White M.F.
      ).
      As skeletal muscle is responsible for over 80% of the peripheral glucose uptake, chronic oxidative stress in this tissue can result in particularly devastating effects on peripheral insulin sensitivity. Exercise is beneficial to patients with metabolic syndrome, and can markedly increase glycemic control (
      • Kayatekin B.M.
      • Gönenç S.
      • Açikgöz O.
      • Uysal N.
      • Dayi A.
      ,
      • Winnick J.J.
      • Sherman W.M.
      • Habash D.L.
      • Stout M.B.
      • Failla M.L.
      • Belury M.A.
      • Schuster D.P.
      ). Exercise stimulates glucose uptake and increases insulin sensitivity in the muscle and other peripheral tissues (
      • Winnick J.J.
      • Sherman W.M.
      • Habash D.L.
      • Stout M.B.
      • Failla M.L.
      • Belury M.A.
      • Schuster D.P.
      ). Muscle contraction during exercise results in elevated oxidative phosphorylation, ROS production, and activation of MAPK cascades, including JNK signaling (
      • Kayatekin B.M.
      • Gönenç S.
      • Açikgöz O.
      • Uysal N.
      • Dayi A.
      ,
      • Kramer H.F.
      • Goodyear L.J.
      ,
      • Aronson D.
      • Boppart M.D.
      • Dufresne S.D.
      • Fielding R.A.
      • Goodyear L.J.
      ,
      • Villa-Caballero L.
      • Nava-Ocampo A.A.
      • Frati-Munari A.C.
      • Rodríguez de León S.M.
      • Becerra-Pérez A.R.
      • Ceja R.M.
      • Campos-Lara M.G.
      • Ponce-Monter H.A.
      ). Paradoxically, exercise-activated JNK does not lead to impaired insulin sensitivity. These contrasting effects of acute oxidative stress during exercise and chronic oxidative stress in metabolic syndrome are not well understood. Indeed, some reports indicate that oxidative stress impairs glucose uptake in the muscle by inhibiting translocation of glucose transporter GLUT4 to plasma membrane (
      • Maddux B.A.
      • See W.
      • Lawrence Jr., J.C.
      • Goldfine A.L.
      • Goldfine I.D.
      • Evans J.L.
      ). Other reports indicate that positive effects of oxidative stress on muscle glucose uptake involve activation of phosphatidylinositol 3-kinase signaling (
      • Kozlovsky N.
      • Rudich A.
      • Potashnik R.
      • Bashan N.
      ,
      • Higaki Y.
      • Mikami T.
      • Fujii N.
      • Hirshman M.F.
      • Koyama K.
      • Seino T.
      • Tanaka K.
      • Goodyear L.J.
      ). It is unclear what mechanistic differences lead to opposite effects of acute and chronic stress on muscle insulin sensitivity and glucose uptake, or why activation of MAPK/JNK signaling causes insulin resistance in the case of chronic, but not acute, oxidative stress.
      To answer these fundamental questions we used a cellular model of muscle insulin resistance based on mouse C2C12 myocytes, which can be made insulin-resistant by culturing in high glucose- and high insulin-containing media, mimicking hyperglycemia and hyperinsulinemia that cause insulin resistance in the pre-diabetic state. We induced acute or chronic stress in insulin-responsive and insulin-resistant myocytes and myotubes to determine their effects on insulin and JNK signaling. Our findings suggest that differential subcellular distributions of JNK-specific phosphatase MKP7 and activated JNK determine the opposite effects of acute and chronic oxidative stress on insulin sensitivity.

      DISCUSSION

      In this study, we evaluated the effects of acute and chronic oxidative stress on insulin and MAPK signaling in regular and insulin-resistant muscle cells. It has been widely established that chronic oxidative stress is detrimental to glucose homeostasis, leading to insulin resistance in muscle and adipose tissue, hyperglycemia, and metabolic disease. Recent work demonstrated that reactive oxygen species play a causative role in the development of insulin resistance and glucose intolerance (
      • Houstis N.
      • Rosen E.D.
      • Lander E.S.
      ). Oxidative stress leads to the activation of MAPK pathways that have been shown by numerous studies to mediate inhibitory serine phosphorylation of IRS1, thereby decreasing the downstream insulin signaling, GLUT4 translocation to the plasma membrane, and glucose uptake (
      • Björnholm M.
      • Zierath J.R.
      ,
      • Evans J.L.
      • Maddux B.A.
      • Goldfine I.D.
      ,
      • Tuncman G.
      • Hirosumi J.
      • Solinas G.
      • Chang L.
      • Karin M.
      • Hotamisligil G.S.
      ). Indeed, animals fed a high-fat diet show increased ROS levels in plasma, increased activation of JNK, and are insulin-resistant. The causality of JNK activation in the development of insulin resistance is demonstrated by the fact that deletion of JNK1 significantly improves insulin sensitivity and glucose homeostasis in high-fat fed mice (
      • Lee Y.H.
      • Giraud J.
      • Davis R.J.
      • White M.F.
      ,
      • Tuncman G.
      • Hirosumi J.
      • Solinas G.
      • Chang L.
      • Karin M.
      • Hotamisligil G.S.
      ,
      • Hotamisligil G.S.
      ,
      • Ozcan U.
      • Cao Q.
      • Yilmaz E.
      • Lee A.H.
      • Iwakoshi N.N.
      • Ozdelen E.
      • Tuncman G.
      • Görgün C.
      • Glimcher L.H.
      • Hotamisligil G.S.
      ). Consistent with the current dogma, we found that chronic oxidative stress caused by either low grade peroxide administration or the hyperglycemia/hyperinsulinemia regimen impairs insulin signaling in two different muscle cell lines. This was due to the reduction in the IRS1 protein level mediated by inhibitory phosphorylation by JNK. JNK activation was essential for the development of insulin resistance in our system, because a specific inhibitor of JNK activation restored insulin-dependent signaling, consistent with the results in db/db and DIO mice, where JNK inhibition improves glucose homeostasis (
      • Kaneto H.
      • Nakatani Y.
      • Miyatsuka T.
      • Kawamori D.
      • Matsuoka T.A.
      • Matsuhisa M.
      • Kajimoto Y.
      • Ichijo H.
      • Yamasaki Y.
      • Hori M.
      ).
      By contrast, we discovered that acute oxidative stress not only did not impair insulin signaling, but increased insulin-dependent AKT phosphorylation and reversed hyperglycemia-induced insulin resistance, restoring insulin stimulation of glucose uptake. Several reports indicate positive effects of oxidative stress on glucose uptake in adipose cells and in isolated muscles. For example, glucose oxidase and peroxide administration has been shown to induce glucose uptake in L6 myotubes (
      • Hirosumi J.
      • Tuncman G.
      • Chang L.
      • Görgün C.Z.
      • Uysal K.T.
      • Maeda K.
      • Karin M.
      • Hotamisligil G.S.
      ). Moreover, oxidative stress induced by either peroxide or xanthine oxidase increases glucose uptake in isolated human muscle, in a phosphatidylinositol 3-kinase-dependent manner (
      • Higaki Y.
      • Mikami T.
      • Fujii N.
      • Hirshman M.F.
      • Koyama K.
      • Seino T.
      • Tanaka K.
      • Goodyear L.J.
      ). Another example of a positive effect of oxidative stress on insulin sensitivity is aerobic exercise. Muscle contraction, which is accompanied by increased oxidative phosphorylation, was shown to significantly increase oxidative stress in human and rodent muscle, followed by activation of MAPK signaling pathways, including JNK (
      • Kramer H.F.
      • Goodyear L.J.
      ,
      • Aronson D.
      • Boppart M.D.
      • Dufresne S.D.
      • Fielding R.A.
      • Goodyear L.J.
      ,
      • Fujii N.
      • Jessen N.
      • Goodyear L.J.
      ). Exercise boosts muscle glucose uptake, in part due to activation of AMP-activated protein kinase signaling, which contributes to GLUT4 translocation in parallel to insulin signaling (
      • Fujii N.
      • Jessen N.
      • Goodyear L.J.
      ). However, exercise also increases insulin sensitivity in the muscle, by an unknown, AMP-activated protein kinase-independent mechanism, and significantly improves glucose tolerance in diabetic humans and animal models, despite increased levels of ROS and MAPK/JNK activation (
      • Winnick J.J.
      • Sherman W.M.
      • Habash D.L.
      • Stout M.B.
      • Failla M.L.
      • Belury M.A.
      • Schuster D.P.
      ). Notably, mice overexpressing the antioxidant gene glutathione surprisingly develop insulin resistance and other metabolic abnormalities, arguing against a simple negative role for ROS and oxidative stress in glucose homeostasis (
      • McClung J.P.
      • Roneker C.A.
      • Mu W.
      • Lisk D.J.
      • Langlais P.
      • Liu F.
      • Lei X.G.
      ). It has recently been proposed that acute oxidative stress, such as that caused by exercise, and chronic metabolic oxidative stress may elicit different cellular responses resulting in contrasting outcomes on insulin sensitivity and glucose homeostasis in the muscle (
      • Higaki Y.
      • Mikami T.
      • Fujii N.
      • Hirshman M.F.
      • Koyama K.
      • Seino T.
      • Tanaka K.
      • Goodyear L.J.
      ). Understanding the mechanisms underlying signaling changes that occur upon acute and chronic stress can be valuable for optimal design of targeted therapies for prevention of insulin resistance.
      We discovered that acute oxidative stress leads to accumulation of activated JNK in the nucleus, whereas chronic oxidative stress, either caused by low grade peroxide administration or hyperglycemia, activates JNK in cytoplasmic pools. This is consistent with the theory that upon chronic metabolic stress JNK phosphorylates IRS1, which is present in the cytoplasm and plasma membrane, to trigger its degradation. Interestingly, acute stress, whereas increasing JNK phosphorylation in the nuclei, significantly decreased activated JNK levels in the cytoplasm of insulin-resistant myoblasts. We found that acute stress led to the exclusion of JNK-specific MKP7 from the nucleus and its accumulation in the cytoplasm. It is plausible that upon acute oxidative stress cytoplasmic JNK is dephosphorylated and inactivated by MKP7. This notion is supported by our finding that MKP7 is required for the increase in insulin sensitivity caused by acute oxidative stress.
      Taken together, our results suggest a novel mechanism of stress-mediated regulation of insulin resistance, where chronic and acute oxidative stresses activate JNK in different subcellular compartments, leading to opposite cellular outcomes. We propose that exercise, similarly to acute oxidative stress, can cause redistribution of MKP7 from the nucleus to the cytoplasm, leading to dephosphorylation of JNK in the cytoplasm and plasma membranes (Fig. 6). This reduction in JNK activation in the cytoplasm and at the plasma membrane should result in increased insulin sensitivity due to IRS1 stabilization, activation of the downstream insulin pathway, and increased glucose uptake (Fig. 6). Our model explains the discrepancies between reported effects of oxidative stress on JNK activation and muscle insulin sensitivity and highlights the importance of differential spatial activation of JNK.
      Figure thumbnail gr6
      FIGURE 6A model for the acute and chronic oxidative stress effects on insulin sensitivity in the muscle. See text for details.

      Acknowledgments

      We thank Akos Szivali for help with confocal imaging, Susan Stevenson, Sandra Souza, Karen Inouye, and Mary Chau for helpful discussions, and Laura Bordone, Kevin Clairmont, and Jesper Gromada for critical reading of this manuscript.

      REFERENCES

        • Demirbag R.
        • Yilmaz R.
        • Gur M.
        • Celik H.
        • Guzel S.
        • Selek S.
        • Kocyigit A.
        Int. J. Clin. Pract. 2006; 60: 1187-1193
        • Rehman A.
        • Nourooz-Zadeh J.
        • Möller W.
        • Tritschler H.
        • Pereira P.
        • Halliwell B.
        FEBS Lett. 1999; 448: 120-122
        • Song F.
        • Jia W.
        • Yao Y.
        • Hu Y.
        • Lei L.
        • Lin J.
        • Sun X.
        • Liu L.
        Clin. Sci. 2007; 112: 599-606
        • Muellenbach E.A.
        • Diehl C.J.
        • Teachey M.K.
        • Lindborg K.A.
        • Archuleta T.L.
        • Harrell N.B.
        • Andersen G.
        • Somoza V.
        • Hasselwander O.
        • Matuschek M.
        • Henriksen E.J.
        Metabolism. 2008; 57: 1465-1472
        • Gao L.
        • Laude K.
        • Cai H.
        Vet. Clin. North Am. Small Animal Pract. 2008; 38: 137-155
        • Björnholm M.
        • Zierath J.R.
        Biochem. Soc. Trans. 2005; 33: 354-357
        • Evans J.L.
        • Maddux B.A.
        • Goldfine I.D.
        Antioxid. Redox Signal. 2005; 7: 1040-1052
        • Houstis N.
        • Rosen E.D.
        • Lander E.S.
        Nature. 2006; 440: 944-948
        • Raman M.
        • Chen W.
        • Cobb M.H.
        Oncogene. 2007; 26: 3100-3112
        • Wang X.
        • Destrument A.
        • Tournier C.
        Biochim. Biophys. Acta. 2007; 1773: 1349-1357
        • Masuda K.
        • Shima H.
        • Watanabe M.
        • Kikuchi K.
        J. Biol. Chem. 2001; 276: 39002-39011
        • Bloch-Damti A.
        • Potashnik R.
        • Gual P.
        • Le Marchand-Brustel Y.
        • Tanti J.D.
        • Rudich A.
        • Bashan N.
        Diabetologia. 2006; 49: 2463-2473
        • Hirosumi J.
        • Tuncman G.
        • Chang L.
        • Görgün C.Z.
        • Uysal K.T.
        • Maeda K.
        • Karin M.
        • Hotamisligil G.S.
        Nature. 2002; 420: 333-336
        • Lee Y.H.
        • Giraud J.
        • Davis R.J.
        • White M.F.
        J. Biol. Chem. 2003; 278: 2896-2902
        • Kayatekin B.M.
        • Gönenç S.
        • Açikgöz O.
        • Uysal N.
        • Dayi A.
        Eur. J. Appl. Physiol. 2002; 87: 141-144
        • Winnick J.J.
        • Sherman W.M.
        • Habash D.L.
        • Stout M.B.
        • Failla M.L.
        • Belury M.A.
        • Schuster D.P.
        J. Clin. Endocrinol. Metab. 2008; 93: 771-778
        • Kramer H.F.
        • Goodyear L.J.
        J. Appl. Physiol. 2007; 103: 388-395
        • Aronson D.
        • Boppart M.D.
        • Dufresne S.D.
        • Fielding R.A.
        • Goodyear L.J.
        Biochem. Biophys. Res. Commun. 1998; 251: 106-110
        • Villa-Caballero L.
        • Nava-Ocampo A.A.
        • Frati-Munari A.C.
        • Rodríguez de León S.M.
        • Becerra-Pérez A.R.
        • Ceja R.M.
        • Campos-Lara M.G.
        • Ponce-Monter H.A.
        Diabetes Res. Clin. Pract. 2007; 75: 285-291
        • Maddux B.A.
        • See W.
        • Lawrence Jr., J.C.
        • Goldfine A.L.
        • Goldfine I.D.
        • Evans J.L.
        Diabetes. 2001; 50: 404-410
        • Kozlovsky N.
        • Rudich A.
        • Potashnik R.
        • Bashan N.
        Free Radical Biol. Med. 1997; 23: 859-869
        • Higaki Y.
        • Mikami T.
        • Fujii N.
        • Hirshman M.F.
        • Koyama K.
        • Seino T.
        • Tanaka K.
        • Goodyear L.J.
        Am. J. Physiol. Endocrinol. Metab. 2008; 294: E889-E897
        • Tuncman G.
        • Hirosumi J.
        • Solinas G.
        • Chang L.
        • Karin M.
        • Hotamisligil G.S.
        Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10741-10746
        • Hotamisligil G.S.
        Diabetes. 2005; 54: S73-S78
        • Ozcan U.
        • Cao Q.
        • Yilmaz E.
        • Lee A.H.
        • Iwakoshi N.N.
        • Ozdelen E.
        • Tuncman G.
        • Görgün C.
        • Glimcher L.H.
        • Hotamisligil G.S.
        Science. 2004; 306: 457-461
        • Kaneto H.
        • Nakatani Y.
        • Miyatsuka T.
        • Kawamori D.
        • Matsuoka T.A.
        • Matsuhisa M.
        • Kajimoto Y.
        • Ichijo H.
        • Yamasaki Y.
        • Hori M.
        Nat. Med. 2004; 10: 1128-1132
        • Fujii N.
        • Jessen N.
        • Goodyear L.J.
        Am. J. Physiol. Endocrinol. Metab. 2006; 291: E867-E877
        • McClung J.P.
        • Roneker C.A.
        • Mu W.
        • Lisk D.J.
        • Langlais P.
        • Liu F.
        • Lei X.G.
        Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 8852-8857